Use of estradiol cypionate as a substitute for GnRH in protocols for synchronizing ovulation in dairy cattle

Our purpose was to determine whether estradiol cypionate (ECP) could be substituted for the second GnRH injection of the standard Ovsynch protocol (injection of GnRH given 7 d before and 48 h after PGF(2alpha), with timed AI [TAI] 12 to 20 h after the second GnRH injection). Lactating dairy cows ranging from 61 to 82 d in milk at TAI were studied in 14 replicates. Main effects were hormone (ECP vs. GnRH) to induce ovulation and exposure to progesterone (P4) or not during the week preceding PGF(2alpha)-induced luteolysis. Four treatments were: 1) 100 microg of GnRH at 48 h after PGF(2alpha) (Ovsynch; n = 27); 2) same as Ovsynch, plus a P4-releasing intravaginal insert (CIDR) placed for 7 d beginning at the first GnRH injection (Ovsynch + CIDR, n = 20); 3) same as Ovsynch, but substituting 1 mg of ECP for GnRH, and injecting ECP at 24 h after PGF(2alpha) (Heatsynch; n = 33); or 4) Heatsynch + CIDR (n = 26). The largest follicle was identified by ultrasonography 24 h after PGF(2alpha) and was monitored every 6 h until ovulation. Incidence of estrus was less after GnRH (54%) than after ECP (87%), but more GnRH-treated cows had LH surges detected (95 vs. 65%) and ovulated (100 vs. 86%). Duration of LH surges, but not peak concentrations, was less after GnRH than after ECP (6.1 +/- 0.7 vs. 12.2 +/- 0.9 h). Pre-treatment with P4 reduced the incidence of LH surges but had no effects on incidence of estrus or ovulation. Intervals to the LH surge and ovulation were less after GnRH than after ECP, but intervals between onset of the LH surge and ovulation did not differ (26 +/- 2 vs. 30 +/- 3 h). We concluded that substituting ECP for GnRH resulted in more cows in estrus and slightly fewer ovulating.     

Evaluation of protocols to synchronize estrus and ovulation in seasonal calving pasture-based dairy production systems

Lactating dairy cows (n = 1,538) were enrolled in a randomized complete block design study to evaluate protocols to synchronize estrus and ovulation. Within each herd (n = 8), cows were divided into 3 calving groups: early, mid, and late, based on days in milk (DIM) at mating start date (MSD). Early calving cows (n = 1,244) were ≥42 DIM at MSD, mid-calving cows (n = 179) were 21 to 41 DIM at MSD, and late-calving cows (n = 115) were 0 to 20 DIM at MSD. Cows in the early, mid-, and late-calving groups were synchronized to facilitate estrus or timed AI (TAI) at MSD (planned breeding 1; PB1), 21 d (PB2), and 42 d (PB3) after MSD, respectively. For each PB, cows in the relevant calving group were stratified by parity and calving date and randomly assigned to 1 of 4 experimental groups: (1) d −10 GnRH (10 μg of i.m. buserelin) and controlled internal drug release insert (CIDR; 1.38 g of progesterone); d −3 PGF_2α (25 mg of i.m. dinoprost); and d −2 CIDR out and AI at observed estrus (CIDR_OBS); (2) same as CIDR_OBS, but GnRH 36 h after CIDR out and TAI 18 h later (CIDR_TAI); (3) same as CIDR_TAI, but no CIDR (Ovsynch); or (4) untreated controls (CTRL). The CIDR_OBS, CIDR_TAI, and Ovsynch had shorter mean intervals from calving to first service compared with the CTRL (69.2, 63.4, and 63.7 vs. 73.7 d, respectively). Both CIDR_OBS (predicted probability; PP of pregnancy = 0.59) and CIDR_TAI (PP of pregnancy = 0.54) had increased odds of conceiving at first service compared with Ovsynch [PP of pregnancy = 0.45; odds ratio (OR) = 1.81 and OR = 1.46, respectively], and Ovsynch had decreased likelihood of conceiving at first service (OR = 0.70) compared with CTRL (PP of pregnancy = 0.53). Both CIDR_TAI hazard ratio; HR [95% confidence interval = 1.21 (1.04, 1.41)] and Ovsynch [HR (95% confidence interval) = 1.23 (1.05, 1.44)] were associated with an increased likelihood of earlier conception compared with the CTRL. A greater proportion of cows on the CIDR_TAI treatment successfully established pregnancy in the first 42 d of the breeding season compared with the CTRL (0.75 vs. 0.67 PP of 42-d pregnancy, respectively). Protocols to synchronize estrus and ovulation were effective at achieving earlier first service and conception in pasture-based seasonal calving dairy herds. However, animals that conceived following insemination at observed estrus had a decreased likelihood of embryo loss to first service compared with animals bred with TAI (PP of embryo loss after first service = 0.05 vs. 0.09; OR = 0.52).     

Pregnancy in dairy cows after synchronized ovulation regimens with or without presynchronization and progesterone

Two experiments examined pregnancy after synchronized ovulation (Ovsynch) with or without progesterone (P4) administered via controlled internal drug release (CIDR) intravaginal inserts. In experiment 1, 262 lactating cows in one herd were in 3 treatments: Ovsynch (n = 91), Ovsynch + CIDR (n = 91), and control (n = 80). The Ovsynch protocol included injections of GnRH 7 d before and 48 h after an injection of PGF20. Timed artificial insemination (TAI; 57 to 77 d postpartum) was 16 to 20 h after the second GnRH injection. Cows in the Ovsynch + CIDR group also received a CIDR (1.9 g of P4) insert for 7 d starting at first GnRH injection. Control cows received A-I when estrus was detected using an electronic estrus detection system. Based on serum P4, 44.1% of cows were cyclic before Ovsynch. Pregnancy rates at 29 d (59.3 vs. 36.3%) and 57 d (45.1 vs. 19.8%) after TAI and embryo survival (75.9 vs. 54.5%) from 29 to 57 d were greater for Ovsynch + CIDR than for Ovsynch alone. In experiment 2, 630 cows in 2 herds received TAI at 59 to 79 d postpartum after 6 treatments. Estrous cycles were either presynchronized (2 injections of PGF2alpha 14 d apart; n = 318) or not presynchronized (n = 312). Within those groups, Ovsynch was initiated 12 d after second presynchronization PGF2alpha, and used alone (n = 318) or with CIDR inserts for 7 d (1.38 g of P4/insert, n = 124 or 1.9 g of P4/insert, n = 188). Before Ovsynch, 80% of cows were cyclic. Presynchronization increased pregnancy (46.8 vs. 37.5%) at 29 d after TAI, but CIDR inserts had no effect on pregnancy in experiment 2. Overall embryonic survival between 29 and 57 d in experiment 2 was 57.7%. Use of CIDR inserts with Ovsynch improved conception and embryo survival in experiment 1 but not in experiment 2, in part due to differing proportions of cyclic cows at the outset. Presynchronization before Ovsynch enhanced pregnancy rate.     

Stage of cycle, incidence, and timing of ovulation, and pregnancy rates in dairy cattle after three timed breeding protocols

In experiment 1, 705 cows were assigned to three treatments: 1) the Ovsynch protocol (a GnRH injection given 7 d before and another 48 h after one PGF2alpha injection); 2) PGF2alpha + Ovsynch (one PGF2alpha injection given 12 d (d -22) before initiating Ovsynch (d -10); and 3) 2xPG12 (two PGF2alpha injections 12 d apart; d -15 and -3, followed 48 h later by a GnRH injection. All cows were inseminated (d 0) 16 to 20 h after the GnRH injection on d -1. Cyclic status was estimated by serum progesterone. More cows were in early diestrus at d -10 for PGF2alpha + Ovsynch (36%) and 2 x PG12 (29%) versus Ovsynch (19%). Multiparous cows receiving PGF2alpha + Ovsynch had greater pregnancy rates via ultrasonography at d 28 after AI (42%) than contemporaries after Ovsynch (28%) or 2xPG12 (27%) but did not differ significantly at palpation 10 to 30 d later (28, 19, and 17%, respectively). Pregnancy of first-parity cows was similar across treatments at 28 d (41%) or at palpation (33%). Pregnancy rates for 128 anestrous cows were lower, regardless of treatment. Overall embryo survival from d 28 until palpation was 72% but was only 44% in 2xPG12 cows that were anestrus through d -10. Experiment 2 included the three treatments above plus controls (one GnRH injection 7 d before PGF2alpha and AI after estrus). Preovulatory follicles were 6 to 11% larger near estrus in controls than on d -1 in cows receiving GnRH. More controls ovulated by 32 h after onset of estrus than were treated cows by 32 h after GnRH, but percentages (79 to 94%) were similar by 40 h. In multiparous cows, PGF2alpha before Ovsynch increased pregnancy rates, whereas the 2xPG12 protocol produced similar pregnancy rates as Ovsynch across parities. Ovulation was effectively induced by 40 h after GnRH.     

Fertility of lactating dairy cows treated with Ovsynch after presynchronization injections of PGF2 alpha and GnRH

Synchronization of ovulation (Ovsynch) using GnRH and PGF2 alpha allows control of follicle growth, corpus luteum regression, and ovulation, but resulting pregnancy rates vary. This study examined whether presynchronization to allow initiation of Ovsynch during diestrus would improve pregnancy rates at timed artificial insemination (AI). Lactating dairy cows (n = 427), 69 to 75 d postpartum, were randomly assigned to two groups by parity. Control cows received Ovsynch (GnRH, d 0; PGF2 alpha, d 7; GnRH, d 9; timed AI 16 h after second GnRH). Treated cows received presynchronization injections of PGF2 alpha and GnRH, 10 and 7 d, respectively, before starting Ovsynch. Pregnancy diagnoses were performed 36 d after AI. Progesterone (P4) concentrations from a subset of cows (n = 84) were determined in serum samples collected on d 0, 3, and 7 of Ovsynch. Presynchronization increased the percentages of cows with > or = 1 ng/ml serum P4 compared with control cows at first injection of GnRH (d 0; 93 vs. 56%) and on d 3 (90.7 vs. 51.2%) during Ovsynch. On day of PGF2 alpha, d 7 during Ovsynch, percentages of cows with > or = 1 ng/ml serum P4 were similar (95.3%, treated vs. 82.9%, control) but more treated cows had > or = 2 ng/ml serum P4 (95.3 vs. 63.4%). However, pregnancy to timed AI was similar between treated (41.5%) and control cows (38.3%). Cows with above-average milk production had greater pregnancy rate (45.8 vs. 33.8%) compared with lower producing cows. Although presynchrony increased the proportion of cows with luteal function at onset of Ovsynch, pregnancy rate to timed AI was not improved. Cows with above-average milk production had greater fertility at timed AI than herdmates with lower milk production.     

A modified presynchronization protocol improves fertility to timed artificial insemination in lactating dairy cows

To compare 2 hormonal protocols for submission of lactating dairy cows for timed artificial insemination (TAI), nonpregnant lactating Holstein cows (n = 269) >60 d in milk were randomly assigned to each of 2 treatments to receive TAI (TAI = d 0). Cows assigned to the first treatment (Ovsynch, n = 134) received 50 microg of GnRH (d -10), 25 mg of PGF2alpha (d -3), and 50 microg of GnRH (d -1) beginning at a random stage of the estrous cycle. Cows assigned to the second treatment (Presynch, n = 135) received Ovsynch but with the addition of 2 PGF2alpha (25 mg) injections administered 14 d apart beginning 28 d (d -38 and -24) before initiation of Ovsynch. All cows received TAI 16 to 18 h after the second GnRH injection. Ovulatory response after each GnRH injection for a subset of cows (n = 109) and pregnancy status 42 d after TAI for all cows were assessed using transrectal ultrasonography. Based on serum progesterone (P4) profiles determined for a subset of cows (n = 109), P4 concentrations decreased for Presynch cows after the first 2 PGF2alpha injections, and Presynch cows had greater P4 concentrations at the PGF2alpha injection on d -3 compared with Ovsynch cows. Although the proportion of cows ovulating after the first and second GnRH injections did not differ statistically between treatments (41.1 and 69.6% vs. 35.9 and 81.1% for Ovsynch vs. Presynch, respectively), pregnancy rate per artificial insemination (PR/AI) at 42 d post TAI was greater for Presynch than for Ovsynch cows (49.6 vs. 37.3%). Parity, DIM, and body condition score (BCS) at TAI did not affect PR/AI to TAI. These data support use of this presynchronization protocol to increase PR/ AI of lactating dairy cows receiving TAI compared with Ovsynch.     

Resynchronizing estrus and ovulation after not-pregnant diagnosis and various ovarian states including cysts

We compared outcomes of 2 protocols used to resynchronize estrus and ovulation in dairy females after a not-pregnant diagnosis. Nulliparous heifers and lactating cows in which artificial insemination (AI) occurred 41 +/- 1 d earlier were presented every 2 to 3 wk for pregnancy diagnosis by using ultrasonography. Ovaries were scanned, follicles were mapped and sized, presence of corpus luteum was noted, and GnRH was injected (d 0). Females were assigned randomly to receive PGF(2alpha) 7 d later (d 7) and then either received estradiol cypionate (ECP) 24 h after PGF(2alpha) (d 8; Heatsynch; n = 230) or a second GnRH injection 48 h after PGF(2alpha) (d 9; Ovsynch; n = 224). Those detected in estrus since their not-pregnant diagnosis were inseminated, whereas the remainder received a timed AI (TAI) between 65 and 74 h after PGF(2alpha). Ovarian scans and blood collected before injections for progesterone analysis were used to classify 4 ovarian status groups: anestrus, follicular cysts, luteal cysts, and cycling, plus an unknown group of females in which no blood sampling or ovarian scans were made. Few females (5.1%) were inseminated between not-pregnant diagnosis and d 8. On d 10, more ECP- than GnRH-treated females were inseminated after detected estrus (24 vs. 6%). Overall, more Ovsynch than Heatsynch females received a TAI (82 vs. 62%). Conception rates tended to be greater for females inseminated after estrus (37%) than after TAI (29%), particularly for those treated with Heatsynch (41 vs. 27%) than with Ovsynch (33 vs. 31%). Those inseminated after estrus conceived 31 +/- 8 d sooner than those receiving the TAI. Conception rates for females having elevated progesterone 7 d after the not-pregnant diagnosis were greater than those having low progesterone in Heatsynch (42%; n = 133 vs. 25%; n = 55) and Ovsynch protocols (33%; n = 142 vs. 15%; n = 45), respectively. Conception rates were greater in nulliparous heifers than in lactating cows (43 vs. 28%) regardless of protocol used. Although overall pregnancy outcomes after a not-pregnant diagnosis were similar in response to either the Ovsynch and Heatsynch protocols, inseminations performed after detected estrus before the scheduled TAI reduced days to eventual conception and tended to increase conception rates, particularly after Heatsynch.     

Effects of gonadotropin-releasing hormone at initiation of the 5-d timed artificial insemination (AI) program and timing of induction of ovulation relative to AI on ovarian dynamics and fertility of dairy heifers

Two experiments evaluated the effects of the first GnRH injection of the 5-d timed artificial insemination (AI) program on ovarian responses and pregnancy per AI (P/AI), and the effect of timing of the final GnRH to induce ovulation relative to AI on P/AI. In experiment 1, 605 Holstein heifers were synchronized for their second insemination and assigned randomly to receive GnRH on study d 0 (n = 298) or to remain as untreated controls (n = 307). Ovaries were scanned on study d 0 and 5. All heifers received a controlled internal drug-release (CIDR) insert containing progesterone on d 0, a single injection of PGF_2α and removal of the CIDR on d 5, and GnRH concurrent with timed AI on d 8. Blood was analyzed for progesterone at AI. Pregnancy was diagnosed on d 32 and 60 after AI. Ovulation on study d 0 was greater for GnRH than control (35.4 vs. 10.6%). Presence of a new corpus luteum (CL) at PGF_2α injection was greater for GnRH than for control (43.1 vs. 20.8%), although the proportion of heifers with a CL at PGF_2α did not differ between treatments and averaged 87.1%. Progesterone on the day of AI was greater for GnRH than control (0.50 ± 0.07 vs. 0.28 ± 0.07 ng/mL). The proportion of heifers at AI with progesterone <0.5 ng/mL was less for GnRH than for control (73.8 vs. 88.2%). The proportion of heifers in estrus at AI did not differ between treatments and averaged 66.8%. Pregnancy per AI was not affected by treatment at d 32 or 60 (GnRH = 52.5 and 49.8% vs. control = 54.1 and 50.0%), and pregnancy loss averaged 6.0%. Responses to GnRH were not influenced by ovarian status on study d 0. In experiment 2, 1,295 heifers were synchronized for their first insemination and assigned randomly to receive a CIDR on d 0, PGF_2α and removal of the CIDR on d 5, and either GnRH 56 h after PGF_2α and AI 16 h later (OVS56, n = 644) or GnRH concurrent with AI 72 h after PGF_2α (COS72; n = 651). Estrus at AI was greater for COS72 than for OVS56 (61.4 vs. 47.5). Treatment did not affect P/AI on d 32 in heifers displaying signs of estrus at AI, but COS72 improved P/AI compared with OVS56 (55.0 vs. 47.6%) in those not in estrus at AI. Similarly, P/AI on d 60 did not differ between treatments for heifers displaying estrus, but CO S72 improved P/AI compared with OVS56 (53.0 vs. 44.7%) in those not in estrus at AI. Administration of GnRH on the first day of the 5-d timed AI program resulted in low ovulation rate and no improvement in P/AI when heifers received a single PGF_2α injection 5 d later. Moreover, extending the proestrus by delaying the final GnRH from 56 to 72 h concurrent with AI benefited fertility of dairy heifers that did not display signs of estrus at insemination following the 5-d timed AI protocol.     

Evaluation of two hormonal protocols for synchronization of ovulation and timed artificial insemination in dairy cows managed in grazing-based dairies

To evaluate the efficacy of two hormonal protocols for synchronization of ovulation and timed artificial insemination (TAI) in dairy cows managed in grazing-based dairies, lactating dairy cows (n = 142) from two grazing-based dairies were randomly assigned to one of three treatment groups. Cows in the first group (Ovsynch) received 50 microg of GnRH (d -10); 25 mg of PGF2alpha (d -3), and 50 microg of GnRH (d -1) followed by timed AI on d 0. Cows in the second group (PGF + Ovsynch) received a modified Ovsynch and timed AI similar to Ovsynch but with the addition of 25 mg of PGF2alpha 12 d (d -22) before initiation of Ovsynch. Cows in the third group (control) received standard reproductive management in place on each farm. Luteolysis occurred in 90.5% of cows exhibiting luteal function on d -22 in the PGF + Ovsynch treatment group, whereas none of the cows in the Ovsynch group underwent luteolysis on d -22. Synchronization rate (i.e., ovulatory response at 48 h after the second GnRH injection), conception rates at TAI and pregnancy rates after 35 d of breeding were similar for cows in the Ovsynch and PGF + Ovsynch groups. The proportion of anovular cows at the first GnRH injection of the synchronization protocols (d -10) was similar for cows receiving Ovsynch (28.0%) and PGF + Ovsynch (30.7%), and conception rate at TAI was similar for cycling (45.8%) and anovular (30.0%) cows. The cumulative pregnancy rate was greater for cows receiving TAI compared with control cows after 7 d of breeding (41.2 vs. 20.0%) but did not differ at 35 d of breeding (54.9 vs. 60.0%). Administration of PGF2alpha 12 d before initiation of Ovsynch did not improve synchronization, conception, or pregnancy rate compared with the standard Ovsynch protocol. Synchronization of ovulation to initiate timed AI at the onset of the breeding season resulted in earlier establishment of pregnancy compared with standard reproductive management.     

Initiation of the breeding season in a grazing-based dairy by synchronization of ovulation

Lactating dairy cows (n = 228) in a semiseasonal, grazing-based dairy were subjected to artificial insemination (AI) to start the 23-d breeding season (d 0 to 22) followed by natural service (d 23 to 120). Cows were randomly assigned to: 1) Ovsynch (GnRH, d -10; PGF2,, d -3; GnRH, d -1; timed AI, d 0) followed by AI at estrus (tail paint removal) on d 1 to 22 (Ovsynch; n = 114); or 2) AI at estrus (tail paint removal) throughout 23 d of AI breeding (tail paint; n = 114). Days to first AI service were greater and the 23-d AI service rate was less for tail paint vs. Ovsynch cows (12.0 +/- 0.6 d vs. 0 d; and 84.2 vs. 100%, respectively). However, conception to first AI was greater for tail paint vs. Ovsynch cows (47.3 vs. 27.3%, respectively). Cows in the tail paint group received only one AI, during 23 d of AI, but 46.4% of Ovsynch cows received a second AI, with similar conception (43.1%) to that of tail paint cows at first AI (47.3%). Based on serum progesterone, incomplete luteal regression after PGF2alpha, and poor ovulatory responses to GnRH contributed to lower conception to timed AI in the Ovsynch group. Cumulative pregnancy rates for tail paint and Ovsynch cows did not differ after 23 d of AI breeding (47.3 vs. 46.3%, respectively) nor after 120 d of AI/ natural service breeding (80.5 vs. 83.3%, respectively). Lactating cows in this grazing-based dairy synchronized poorly to Ovsynch resulting in reduced conception to timed AI compared with AI after tail paint removal.     

Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with Ovsynch initiated 32 d after timed artificial insemination

The objective was to determine if using a Double-Ovsynch protocol [DO; Pre-Resynch: GnRH-7 d-PGF(2α)-3 d-GnRH, 7 d later Breeding-Resynch: GnRH-7 d-PGF(2α)-56 h-GnRH-16 h-timed artificial insemination (TAI)] to resynchronize ovulation after a previous TAI would increase synchrony and pregnancies per AI (P/AI) compared with an Ovsynch protocol initiated 32 d after TAI (D32; GnRH-7 d-PGF(2α)-56 h-GnRH-16 h-TAI). Lactating Holstein cows at various days in milk and prior AI services were blocked by parity and randomly assigned to resynchronization treatments. All DO cows received the first GnRH injection of Pre-Resynch 22 d after TAI, and cows (n=981) diagnosed not pregnant using transrectal ultrasonography 29 d after TAI continued the protocol. Pregnancy status for all D32 cows was evaluated 29 d after TAI so fertility and pregnancy loss could be compared with that of DO cows. All D32 cows received the first GnRH injection of Ovsynch 32 d after TAI, and cows (n=956) diagnosed not pregnant using transrectal palpation 39 d after TAI continued the protocol. In a subgroup of cows from each treatment, ultrasonography (n=751) and serum progesterone (P4) concentrations (n=743) were used to determine the presence of a functional corpus luteum (CL) and ovulation to the first GnRH injection of D32 and Breeding-Resynch of DO (GnRH1), luteal regression after PGF before TAI, and ovulation to the GnRH injection before TAI (GnRH2). Overall, P/AI 29 d after TAI was not affected by parity and was greater for DO compared with D32 cows (39 vs. 30%). Pregnancy loss from 29 to 74 d after TAI was not affected by parity or treatment. The percentage of cows with a functional CL (P4 ≥1.0 ng/mL) at GnRH1 was greater for DO than D32 cows (81 vs. 58%), with most DO cows having medium P4 (60%; 1.0 to 3.49 ng/ml), whereas most D32 cows had either low (42%; <1.0 ng/mL) or high (36%; ≥3.5 ng/mL) P4 at GnRH1. Ovulation to GnRH1 was similar between treatments but was affected by serum P4 at GnRH. Cows with low P4 (<1.0 ng/mL) had the greatest ovulatory response (59%), followed by cows with medium (≥1.0 to 3.49 ng/mL; 38%) and then high (≥3.50 ng/mL; 16%) P4 at GnRH1. A greater percentage of DO cows were synchronized compared with D32 cows (72 vs. 51%) primarily due to a greater percentage of D32 than DO cows without a functional CL at the PGF injection before TAI (35 vs. 17%) or without complete CL regression before GnRH2 (17 vs. 7%). We conclude that DO increased fertility of lactating dairy cows during a resynchronization program primarily by increasing synchronization of cows during the Ovsynch protocol before TAI.     

Pregnancy rates after timed AI of heifers following removal of intravaginal progesterone inserts

Reproductive performance of dairy heifers was compared for each of 2 synchronization protocols: The first group of 54 heifers was synchronized using intravaginal progesterone inserts (CIDR) plus estradiol cypionate (ECP) on d 0, PGF(2alpha) on d 7, and ECP again on d 8 (CIDR-ECP); a second group of 56 heifers was synchronized using CIDR and ECP on d 0, PGF(2alpha) on d 7, and GnRH on d 9 (CIDR-GnRH). All heifers received timed artificial insemination (TAI) at 48, 56, or 72 h after CIDR removal on d 7. Pregnancy diagnosis was conducted by ultrasonography 32 +/- 1 d post AI to confirm pregnancy and at 60 +/- 1 d post AI to determine embryo survival. Ovaries were monitored by ultrasonography daily from d 0 to 7 and twice daily from d 8 to ovulation to examine emergence of a new wave of follicles, size of the ovulatory follicle, and timing of ovulation on 15 heifers per protocol. New follicular development was detected 3.7 +/- 0.2 d after CIDR insertion. Heifers receiving CIDR-ECP had a shorter interval from CIDR removal to ovulation than heifers receiving CIDR-GnRH (63.8 +/- 3.0 vs. 71.6 +/- 2.3 h, respectively); however, ovulation occurred 39.8 +/- 3.0 h after ECP or 23.6 +/- 2.3 h after GnRH. Diameters of ovulatory follicles did not differ between treatments. Overall pregnancy rate for synchronized heifers was 60.1%, and embryo survival was 98%. Pregnancy rate for heifers synchronized with CIDR-ECP was 63.0% and similar to that in heifers synchronized with CIDR-GnRH (57.1%). Pregnancy rate was affected by time of AI for heifers synchronized using CIDR-ECP but not for those synchronized with CIDR-GnRH. Heifers in the CIDR-ECP group that were inseminated 56 h after CIDR removal had a higher pregnancy rate (81.0%) compared with heifers inseminated 48 (66.7%) or 72 h (50.0%) after CIDR removal. Either ECP or GnRH used in a CIDR-based TAI program in dairy heifers can achieve acceptable reproductive performance.     

Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature

Two treatments designed to increase circulating progesterone concentration (P4) during preovulatory follicle development were compared. One treatment used 2 intravaginal P4 implants (controlled internal drug-releasing inserts; CIDR) and the other used a GnRH treatment at beginning of the protocol. Lactating Holstein cows that had been diagnosed as nonpregnant were randomly assigned to receive timed artificial insemination (TAI) following 1 of 2 treatments (n = 1,638 breedings): (1) GnRH: CIDR+ 2 mg of estradiol (E2) benzoate + 100 µg of GnRH on d ?11, PGF_2α on d ?4, CIDR withdrawal + 1.0 mg of E2-cypionate + PGF_2α) on d ?2, and TAI on d 0; or (2) 2CIDR: 2 CIDR + 2 mg of E2-benzoate on d ?11, 1 CIDR withdrawn + PGF_2α on d ?4, second CIDR withdrawn + 1.0 mg of E2-cypionate + PGF_2α on d ?2, and TAI on d 0. Milk yield was measured daily between d 0 and d 7. Rectal temperature was measured using a digital thermometer at d 0 and 7, and elevated body temperature was defined as an average rectal temperature ≥39.1°C. Pregnancy diagnoses were performed on d 32 and 60 after TAI. We detected no effect of treatments on pregnancy per AI or pregnancy loss regardless of elevated body temperature, body condition score, parity, milk yield, or presence or absence of a corpus luteum (CL) on d ?11 or d ?4. Pregnancy per AI at 60 d was reduced [elevated body temperature = 22.8% (162/709), no elevated body temperature 34.1% (279/817)] and pregnancy loss tended to increase [elevated body temperature = 20.2% (41/203), no elevated body temperature 14.4% (47/326)] in cows with elevated body temperature. Various physiological measurements associated with greater fertility were also reduced in cows with elevated body temperature, such as percentage of cows with a CL at PGF_2α (decreased 7.9%), ovulatory follicle diameter (decreased 0.51 mm), expression of estrus (decreased 5.1%), and ovulation near TAI (decreased 2.8%) compared with cows without elevated body temperature. A greater proportion of cows (30.2%) had a CL at PGF_2α in the GnRH treatment [74.1% (570/763)] than in the 2CIDR treatment [56.9% (434/763)]; however, circulating P4 concentration was greater at the time of PGF_2α treatment (d ?4) for cows 2CIDR (4.26 ± 0.13 ng/mL) than in cows in GnRH (3.99 ± 0.14 ng/mL). Thus, these 2 protocols yield similar fertility results that might be due to somewhat different physiological alterations. Treatment with GnRH increased the proportion of cows with a CL at PGF_2α; however, the 2CIDR protocol increased circulating P4 under all circumstances.     

Follicular wave of the ovulatory follicle and not cyclic status influences fertility of dairy cows

Two experiments evaluated the influence of follicular wave at artificial insemination (AI) on fertility of dairy cows. In experiment 1, data from 5,607 lactating cows enrolled in estrous and ovulation synchronization programs for AI were evaluated. Cows’ blood was analyzed for progesterone 7 to 14 d apart, with the second sample collected on the day of the first GnRH (GnRH1) of the synchronization protocol. Cows were classified as cyclic if progesterone was ≥1 ng/mL in at least 1 of the 2 samples and as anovular if both samples were <1 ng/mL. Cyclic cows were categorized as low (CLOW; < 1 ng/mL) or high (CHIGH; ≥ 1 ng/mL) progesterone on the day of GnRH1, which would result in ovulation of the dominant follicle of the first (FW) and second (SW) follicular waves, respectively, at AI. Pregnancy per AI (P/AI) was determined 30 and 53 d after AI. In experiment 2, 220 cyclic Holstein cows received 2 injections of PGF_2α administered 14 d apart. The Ovsynch protocol (d 0 GnRH, d 7 PGF_2α, d 9 GnRH, d 9.5 timed AI) was initiated either 3 or 10 d after the second PGF_2α of the presynchronization to result in insemination to the FW or SW dominant follicles. Blood was analyzed for progesterone and ovaries were scanned to determine ovulatory responses and follicle diameter. Pregnancy was determined on d 32 and 67 after timed AI. In experiment 1, P/AI on d 30 was greater for CHIGH cows than for CLOW and anovular cows (43.0, 31.3, and 29.7%, respectively), but because of pregnancy loss, P/AI on d 53 was lowest for anovular cows. Proportions of cows with short reinsemination intervals differed among groups and were 7.1, 15.7, and 11.9% for CHIGH, CLOW, and anovular cows, respectively. Pregnancy loss was greater for anovular cows than for CLOW cows (15.0 vs. 10.0%) and was intermediate for CHIGH cows (13.5%). In experiment 2, 9.8 and 97.2% of the FW and SW cows, respectively, had progesterone ≥1 ng/mL at GnRH1. Concentrations of progesterone at the GnRH1 and PGF_2α injections of the Ovsynch protocol were greater for SW cows than FW cows. Pregnancy per AI was greater for SW cows than for FW cows (41.7 vs. 30.4%) despite less ovulation to GnRH1 in SW cows than in FW cows (78.7 vs. 88.4%). Collectively, these data indicate that follicular wave of the ovulatory follicle and not cyclic status caused the greatest reduction in P/AI in dairy cows. Whether the culprit is the follicle itself or the hormonal milieu characteristic of the first follicular wave and the early stage of the estrous cycle remains to be elucidated. Synchronization programs that induced ovulation of the FW follicle at AI reduced P/AI in lactating dairy cows, and ovulation of the FW follicle, or development of the ovulatory follicle under low progesterone concentrations, or both, might be mechanisms for reduced fertility in anovular cows.     

Use of intravaginal progesterone-releasing inserts in a synchronization protocol before timed AI and for synchronizing return to estrus in Holstein heifers

Holstein heifers (n = 189) were submitted to a 42-d artificial insemination (AI) period in which they underwent AI after once-daily evaluation of rubbed tail chalk. At the onset of the AI period (d 0), heifers were assigned randomly to receive synchronization of ovulation and timed AI (TAI; d 0: 100 mug of GnRH; d 6: 25 mg of PGF(2alpha); d 8: 100 mug of GnRH + TAI) either without (GPG; n = 95), or with inclusion of a CIDR insert (CIDR; n = 94) from d 0 to 6. No CIDR heifers received AI before d 8 compared with 24% of GPG heifers, and pregnancy rate per AI (PR/AI) at 30 d after TAI did not differ between treatments. To synchronize return to estrus for heifers failing to conceive after TAI, heifers (n = 166) receiving TAI to first service were randomly assigned to receive no further treatment (control; n = 85) or receive a new CIDR insert between 14 and 20 d after TAI (Resynch; n = 81). No Resynch heifers received AI during CIDR treatment compared with 35% of control heifers, and the proportion of heifers receiving AI within 72 h after the day of CIDR removal was 78 vs. 50% for Resynch vs. control heifers, respectively. No treatment x inseminator interaction was detected at first or second AI; however, overall PR/AI was modest for heifers throughout the experiment due to poor performance of 2 of the 3 herd inseminators (14, 6, and 58% PR/AI, respectively). Inclusion of CIDR inserts suppressed estrus during the TAI protocol with no reduction in PR/ AI. Resynchronization of estrus using CIDR inserts resulted in tighter synchrony of estrus among nonpregnant heifers compared with untreated controls.     

Progesterone, Follicular, and Estrual Responses to Progesterone-Based Estrus and Ovulation Synchronization Protocols at Five Stages of the Estrous Cycle

The objective was to monitor changes in ovarian status in heifers exposed to a progesterone insert with or without concurrent GnRH injection. Estrus was manipulated in 283 heifers (31 breeding clusters) by administering GnRH, progesterone, and PGF_2α at 5 stages of the estrous cycle. Estrus was presynchronized with a progesterone insert (CIDR) for 7 d before PGF_2α was administered 24 h before insert removal. Successive clusters of heifers were assigned to treatments (2 heifers per treatment) on cycle d 2, 5, 10, 15, and 18. Treatments consisted of a progesterone insert (d 0) for 7 d plus: 1) PGF_2α on d 6, 24 h before insert removal (early PGF); 2) GnRH on d 0 + early PGF_2α (GnRH + early PGF); 3) PGF_2α at insert removal (late PGF); and 4) GnRH on d 0 + late PGF (GnRH + late PGF). Controls received GnRH on d 0 and PGF_2α on d 7. Ovaries were scanned by transrectal ultrasonography on d 0, 2, 7, 9, and 11 to assess follicle diameters and ovulation. Blood was collected on d 0, 2, 6, 7, 8, and 9 to quantify serum concentrations of progesterone. Insemination occurred after detected estrus or by timed artificial insemination (TAI) at 64 h after insert removal. Only 25% of 141 GnRH-treated heifers ovulated by d 2; twice as many ovulated when treatment was initiated on d 5 (46.4%) than on other cycle days (20.3%). Diameters of the largest follicle exposed to GnRH on d 0, 2, 7, or 9 did not differ regardless of whether ovulation occurred. Small treatment and stage of cycle differences in diameter of the largest follicle were detected on d 2, 7, and 9. Compared with controls, progesterone concentration was greater in all progesterone-treated heifers on d 2 and 6. Early- vs. late-PGF treatment resulted in less progesterone on d 7 and 8. Pregnancies per AI were less after TAI (44%) than after detected estrus (56%) and were less in controls than in all progesterone treatments. Heifers in which treatments were initiated on d 10 of the cycle had the most consistent (estrus vs. TAI) pregnancies per AI (65.4%) compared with other cycle days. Compared with controls, more progesterone-treated heifers ovulated by 96 h after insert removal. Application of the progesterone insert reduced variance of the interval to estrus after insert removal (or PGF_2α injection in controls) by 1.6-fold compared with controls. These results do not support the use of GnRH in a progesterone-based synchronization protocol.     

Use of estradiol cypionate in a presynchronized timed artificial insemination program for lactating dairy cattle

Experiment 1 evaluated pregnancy rates when estradiol cypionate (ECP) was used to induce ovulation as part of a timed artificial insemination (TAI) protocol in comparison to Ovsynch for lactating dairy cows in Florida (n = 371) and Texas (n = 321). Cows were presynchronized with two injections of PGF2, (25 mg, im) given 14 d apart with TAI protocols beginning 14 d after the second injection of PGF20. The TAI protocols consisted of an injection of GnRH (100 microg, im) followed by PGF2alpha 7 d later. Then, cows either received an injection of GnRH (Treatment I, Ovsynch) at 48 h after PGF2alpha and inseminated 16 to 24 h later or received an injection of ECP (1 mg, i.m.) at 24 h after PGF2alpha, (Treatment II; Heatsynch) and inseminated 48 h later. In Florida, pregnancy rates after TAI were 37.1 +/- 5.8% for Ovsynch compared with 35.1 +/- 5.0% for Heatsynch. In Texas, pregnancy rates were 28.2 +/- 3.6% for Ovsynch and 29.0 +/- 3.5% for Heatsynch. Overall pregnancy rates did not differ between Ovsynch and Heatsynch treatments. In Experiment 2, estrus and ovulation times were determined in lactating dairy cows submitted to the Heatsynch protocol. Frequencies of detected estrus and ovulation after ECP were 75.7% (28/37) and 86.5% (32/37), respectively. Mean intervals to ovulation were 55.4 +/- 2.7 h (n = 32) after ECP and 27.5 +/- 1.1 h (n = 27) after onset of estrus. Estrus occurred at 29.0 +/- 1.8 h (n = 28) after ECP. It is recommended that any cow detected in estrus by 24 h after ECP injection be inseminated at 24 h and all remaining cows be inseminated at 48 h because 75% (n = 24/32) of the ovulations occurred between > or = 48 h to < or = 72 h after ECP. Synchronization of ovulation and subsequent fertility indicated that estradiol cypionate could be used to induce ovulation for successful timed insemination.     

Supplementation with estradiol-17beta before the last gonadotropin-releasing hormone injection of the Ovsynch protocol in lactating dairy cows

The aim of this study was to determine whether an increase in circulating estrogen concentrations would increase percentage pregnant per artificial insemination (PP/AI) in a timed AI protocol in high-producing lactating dairy cows. We analyzed only cows having a synchronized ovulation to the last GnRH of the Ovsynch protocol (867/1,084). The control group (n = 420) received Ovsynch (GnRH - 7 d - PGF_2α - 56 h - GnRH - 16 h - timed AI). The treatment group (n = 447) had the same timed AI protocol with the addition of 1 mg of estradiol-17β (E2) at 8 h before the second GnRH injection. Ovarian ultrasound and blood samples were taken just before E2 treatment of both groups. In a subset of cows (n = 563), pressure-activated estrus detection devices were used to assess expression of estrus at 48 to 72 h after PGF_2α treatment. Ovulation was confirmed by ultrasound 7 d after timed AI. Treatment with E2 increased expression of estrus but overall PP/AI did not differ between E2 and control cows. There was an interaction between treatment and expression of estrus such that PP/AI was greater in E2-treated cows that showed estrus than in E2-treated or control cows that did not show estrus and tended to be greater than control cows that showed estrus. There was evidence for a treatment by ovulatory follicle size interaction on PP/AI. Supplementation with E2 improved PP/AI in cows ovulating medium (15 to 19 mm) but not smaller or larger follicles. The E2 treatment also tended to improve PP/AI in primiparous cows with low (≤2.5) body condition score, and in cows at first postpartum service compared with Ovsynch alone. In conclusion, any improvements in PP/AI because of E2 treatment during a timed AI protocol appear to depend on expression of estrus, parity, body condition score, and size of ovulatory follicle.     

Resynchronization of ovulation protocols for dairy cows including or not including gonadotropin-releasing hormone to induce a new follicular wave: Effects on re-insemination pattern,ovarian responses,and pregnancy outcomes

Our objectives were to evaluate the pattern of re-insemination, ovarian responses, and pregnancy per artificial insemination (P/AI) of cows submitted to different resynchronization of ovulation protocols. The base protocol started at 25 ± 3 d after artificial insemination (AI) and was as follows: GnRH, 7 and 8 d later PGF_2α, GnRH 32 h after second PGF_2α, and fixed timed AI (TAI) 16 to 18 h after GnRH. At 18 ± 3 d after AI, cows were randomly assigned to the G25 (n = 1,100) or NoG25 (n = 1,098) treatments. The protocol for G25 and NoG25 was the same, except that cows in NoG25 did not receive GnRH 25 ± 3 d after AI. At nonpregnancy diagnosis (NPD), 32 ± 3 d after AI, cows from G25 and NoG25 with a corpus luteum (CL) ≥15 mm in diameter and a follicle ≥10 mm completed the protocol (G25 CL = 272, NoG25 CL = 194), whereas cows from both treatments that did not meet these criteria received a modified Ovsynch protocol with P4 supplementation [controlled internal drug release insert plus GnRH, controlled internal drug release insert removal, and PGF_2α 7 and 8 d later, GnRH 32 h after second PGF_2α, and TAI 16 to 18 h after GnRH (G25 NoCL = 53, NoG25 NoCL = 78)]. Serum concentrations of progesterone (P4) were determined and ovarian ultrasonography was performed thrice weekly from 18 ± 3 d after AI until 1 d after TAI (G25 = 46, NoG25 = 44 cows). A greater percentage of NoG25 cows were re-inseminated at detected estrus (NoG25 = 53.5%, G25 = 44.6%), whereas more cows had a CL at NPD in G25 than NoG25 (83.7 and 71.3%). At 32 d after AI, P/AI was similar for G25 and NoG25 for inseminations at detected estrus (38.4 and 42.9%), TAI services for cows with no CL (40.4 and 36.7%), and for all services combined (39.6 and 39.0%). However, P/AI were greater for cows with a CL in G25 than NoG25 (40.6 and 32.8%) that received TAI. More cows ovulated spontaneously or in response to GnRH for the G25 than the NoG25 treatment (70 and 36%) but a similar proportion had an active follicle at NPD (G25 = 91% and NoG25 = 96%). The largest follicle diameter at NPD (G25 = 15.0 ± 0.4 mm, NoG25 = 16.5 ± 0.6 mm) and days since it reached ≥10 mm (G25 = 4.0 ± 0.3 d, NoG25 = 5.8 ± 0.6 d) were greater for the NoG25 than G25 treatment. For cows with a CL at NPD, CL regression after NPD, ovulation after TAI, and ovulatory follicle diameter did not differ. In conclusion, removing the first GnRH of a modified Resynch-25 protocol for cows with a CL at NPD and a modified Ovsynch protocol with P4 supplementation for cows without a CL at NPD resulted in a greater percentage of cows re-inseminated at detected estrus and a similar proportion of cows pregnant in spite of reduced P/AI for cows with a CL at NPD.     

Treatment of cycling and noncycling lactating dairy cows with progesterone during Ovsynch

Our objective was to determine whether progesterone (P4) supplementation during an Ovsynch protocol would enhance fertility in lactating dairy cows. Lactating dairy cows (n = 634) at 6 locations were assigned randomly within lactation number and stage of lactation to receive the Ovsynch protocol [OVS; synchronization of ovulation by injecting GnRH 7 d before and 48 h after PGF_2α, followed by one fixed-time AI (TAI) 16 to 20 h after the second GnRH injection] or Ovsynch plus a controlled internal drug release (CIDR) P4-releasing insert for 7 d, beginning at the first GnRH injection (OVS + CIDR). Blood was sampled to quantify P4 10 d before the first GnRH injection, immediately before the first GnRH injection, at the time of CIDR removal, before the PGF_2α injection (1 to 2 h after CIDR insert removal), and 48 h after the PGF_2α injection to determine cyclicity status before initiation of treatment, luteal status at the PGF_2α injection, and incidence of luteal regression. Overall, conception rates at 28 (40 vs. 50%) and 56 d (33 vs. 38%) after TAI differed between OVS and OVS + CIDR, respectively; but a treatment × location interaction was detected. Compared with OVS, pregnancy outcomes were more positive for OVS + CIDR cows at 4 of 6 locations 28 d after TAI and at 3 of 6 locations 56 d after TAI. An interaction of luteal status (high vs. low) before CIDR insert removal and PGF_2α injection with pretreatment cycling status indicated that cows having low P4 at PGF_2α injection benefited most from P4 supplementation (OVS + CIDR = 36% vs. OVS = 18%), regardless of pretreatment cycling status. Pregnancy loss between 28 and 56 d after TAI was greater for noncycling cows (31%) compared with cycling cows (16%). Pregnancy loss for cows receiving P4 (21%) did not differ from that for cows not receiving P4 (21%). Supplementation of P4, pretreatment cycling status, and luteal status before PGF_2α injection altered follicular diameters at the time of the second GnRH injection, but were unrelated to pregnancy outcomes. Incidence of multiple ovulation was greater in noncycling than in cycling cows. Further, cows having multiple ovulations had improved pregnancy outcomes at 28 and 56 d after TAI. In summary, a CIDR insert during the Ovsynch protocol increased fertility in lactating cows having low serum P4 before PGF_2α injection. Improved pregnancy outcomes were observed at some, but not all locations.